Oxidant stress and phospholipase A₂ (PLA₂) activation have been implicated in numerous proinflammatory responses of the mesangial cell (MC). We investigated the cross-talk between group IVα cytosolic PLA₂ (cPLA₂α) and secretory PLA₂s (sPLA₂s) during H₂O₂-induced arachidonic acid (AA) release using two types of murine MC: (i) MC^+/+, which lack group IIa and V PLA₂s, and (ii) MC^–/–, which lack groups IIa, V, and IVα PLA₂s. H₂O₂-induced AA release was greater in MC^+/+ compared with MC^–/–. It has been argued that cPLA₂α plays a regulatory role enhancing the activity of sPLA₂s, which act on phospholipids to release fatty acid. Group IIa, V, or IVα PLA₂s were expressed in MC^–/– or MC^+/+ using recombinant adenovirus vectors. Expression of cPLA₂α in H₂O₂-treated MC^–/– increased AA release to a level approaching that of H₂O₂-treated MC^+/+. Expression of either group IIa PLA₂ or V PLA₂ enhanced AA release in MC^+/+ but had no effect on AA release in MC^–/–. When sPLA₂ and cPLA₂α are both present, the effect of H₂O₂ is manifested by preferential release of AA compared with oleic acid. Inhibition of the ERK and protein kinase C signaling pathways with the MEK-1 inhibitor, U0126, and protein kinase C inhibitor, GF 1092030x, respectively, and chelating intracellular free calcium with 1,2-bis(2-aminophenoyl)ethane-N,N,N′,N′-tetraacetic acid-AM, which also reduced ERK1/2 activation, significantly reduced H₂O₂-induced AA release in MC^+/+ expressing either group IIa or V PLA₂s. By contrast, H₂O₂-induced AA release was not enhanced when ERK1/2 was activated by infection of MC^+/+ with constitutively active MEK1-DD. We conclude that the effect of group IIa and V PLA₂s on H₂O₂-induced AA release is dependent upon the presence of cPLA₂α and the activation of PKC and ERK1/2. Group IIa and V PLA₂s are regulatory and cPLA₂α is responsible for AA release.